Extracellular reduction of via conductive pili as a protective cellular mechanism

Dena L. Cologgia, Sanela Lampa-Pastirka, Allison M. Speersa, Shelly D. Kellyb, and Gemma Regueraa,1

aDepartment of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824; and bExtended X-ray Absorption Fine Structure (EXAFS) Analysis, Bolingbrook, IL 60440

Edited by François M. Morel, Princeton University, Princeton, NJ, and approved August 5, 2011 (received for review May 31, 2011)

The in situ stimulation of Fe(III) oxide reduction by Geobacter bac- sulfurreducens (11) motivated molecular studies to elucidate the teria leads to the concomitant precipitation of hexavalent uranium biological mechanism behind this reaction. Because c-cytochromes [U(VI)] from groundwater. Despite its promise for the bioremedia- are abundant in the cell envelope of Geobacter , studies tion of uranium contaminants, the biological mechanism behind focused on identifying extracytoplasmic c-cytochromes that could this reaction remains elusive. Because Fe(III) oxide reduction re- function as dedicated U reductases (12, 13). However, mutations quires the expression of Geobacter’s conductive pili, we evaluated were often pleitrophic (14–16) and showed either no defect or only their contribution to uranium reduction in Geobacter sulfurredu- partial defects in the cell’s ability to remove U(VI) (12, 13). In- cens grown under pili-inducing or noninducing conditions. A pilin- terpretation was also difficult due to inconsistencies in the repor- deficient mutant and a genetically complemented strain with ted mutant phenotypes, with some mutations reportedly abolishing reduced outer membrane c-cytochrome content were used as con- U(VI) removal activities, yet mutant cells showing extensive trols. Pili expression significantly enhanced the rate and extent of mineralization (13). Furthermore, these studies consistently uranium immobilization per cell and prevented periplasmic miner- showed that the U precipitated inside the cell envelope. U is not alization. As a result, pili expression also preserved the vital respi- known to be essential for the synthesis of any cell component or for ratory activities of the cell envelope and the cell’s viability. Uranium any cellular biological reaction, yet can be reduced and pre- fi preferentially precipitated along the pili and, to a lesser extent, on cipitated nonspeci cally by the abundant low-potential electron donors of the cell envelope of Gram-negative bacteria (17). This is outer membrane -active foci. In contrast, the pilus-defective SCIENCES predicted to compromise the integrity of the cell envelope and its

strains had different degrees of periplasmic mineralization match- ENVIRONMENTAL ing well with their outer membrane c-cytochrome content. X-ray vital functions (18). Because of this, the environmental relevance of these early studies in G. sulfurreducens is questionable. absorption spectroscopy analyses demonstrated the extracellular Geobacter reduction of U(VI) by the pili to mononuclear tetravalent uranium The energy to support the growth of bacteria after in situ stimulation results from the reduction of the abundant Fe(III) U(IV) complexed by carbon-containing ligands, consistent with a bi- oxides, a process that requires the expression of their conductive ological reduction. In contrast, the U(IV) in the pilin-deficient mutant pili (19). In contrast to the lack of conservation of c-cytochrome cells also required an additional phosphorous ligand, in agreement sequences (20), the genes encoding the Geobacteraceae pilus sub- with the predominantly periplasmic mineralization of uranium ob- units or pilins are highly conserved and form an independent line of served in this strain. These findings demonstrate a previously un- ’ Geobacter descent (19). This is consistent with the pili s specialized function as recognized role for conductive pili in the extracellular electrical conduits. The pilus apparatus is anchored in the cell en- reduction of uranium, and highlight its essential function as a cata- velope of Gram-negative cells (21) and could potentially accept lytic and protective cellular mechanism that is of interest for the electrons from cell envelope c-cytochromes or the menaquinone of uranium-contaminated groundwater. pool in the inner membrane. Pili also protrude outside the cell envelope and can reach micrometer lengths, thereby enhancing the biological electron transfer | pilus nanowires | Geobacteraceae | metal cell’s reactive surface. Thus, we hypothesized that the pili could reduction catalyze the reduction of U(VI) “at a distance” to maximize the cell’s catalytic surface while minimizing exposure to U. Here we issimilatory metal-reducing microorganisms gain energy for show that the conductive pili of G. sulfurreducens catalyze the ex- Dgrowth by coupling the oxidation of organic acids or H2 to the tracellular reduction of U(VI) to a mononuclear U(IV) phase and reduction of metals. Some can also use uranium (U) as an elec- prevent its periplasmic mineralization. This mechanism preserves tron acceptor (1, 2), a process that could be harnessed for the the functioning and integrity of the cell envelope and the cell’svi- bioremediation of the contaminated aquifers and sediments ability. These results demonstrate that pili are the elusive U re- left by the intensive U mining practices of the Cold War era (3). ductase of Geobacter bacteria and that their catalytic function also Interestingly, stimulating the activity of metal-reducing micro- serves as a protective cellular mechanism. Our findings suggest that organisms in situ resulted in the concomitant removal of soluble pili’s expression confers on Geobacter bacteria an adaptive eco- hexavalent uranium [U(VI)] from the contaminated groundwater logical advantage in the contaminated subsurface of potential in- and detection of its sparingly soluble, less mobile form, tetravalent terest for the optimization of in situ bioremediation. uranium [U(IV)] in sediments (4–8). This suggests that stimu- lating metal reduction in the subsurface results in the biological Results reduction of U(VI) to U(IV), thereby preventing plume migration Expression of Pili Promotes the Extracellular Reduction of U(VI). The and eliminating the potential for contaminant exposure. correspondence between pili expression and U immobilization The removal of U(VI) from groundwater following the in situ stimulation of metal reduction is often concomitant with sub- stantial increases in the growth and activity of dissimilarity metal- Author contributions: D.L.C. and G.R. designed research; D.L.C., S.L.-P., A.M.S., and S.D.K. reducing microorganisms in the family Geobacteraceae (4, 6, 7, 9). performed research; D.L.C., S.D.K., and G.R. analyzed data; and S.D.K. and G.R. wrote Despite extensive efforts to understand the mechanisms and the paper. pathways used by these bacteria to reduce U(VI), the nature of its The authors declare no conflict of interest. U reductase has remained elusive for almost two decades. Early This article is a PNAS Direct Submission. studies with Geobacter metallireducens GS15 suggested that U was 1To whom correspondence should be addressed. E-mail: [email protected]. reduced extracellularly to uraninite under conditions conducive to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cell growth (1, 10). The development of genetic tools in Geobacter 1073/pnas.1108616108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1108616108 PNAS Early Edition | 1of5 Downloaded by guest on October 3, 2021 was examined by monitoring the removal of U(VI) acetate from outer membrane c-cytochromes also contribute to the extracel- solution by resting wild-type cells incubated at 25 °C (WTP+)or lular reduction of U(VI). It is unlikely that the outer membrane 30 °C (WT −) to induce or prevent pili assembly, respectively. c-cytochrome OmcS, which has been hypothesized to mediate P − Controls with a pilin-deficient mutant (PilA ) and its genetically electron transfer between the conductive pili and metals (22), complemented strain (pRG5::pilA) were also included. The pili- contributed to the pili-mediated reduction of U, because the ::pilA ated strains WTP+ and pRG5::pilA removed substantially more pRG5 strain expressed OmcS at wild-type levels (Fig. S5) yet − A U(VI) from solution than the nonpiliated strains WTP− and PilA reduced more U than the WTP+ (Fig. 1 ) and proportionally to (Fig. 1A). The activity was biological in nature, inasmuch as heat- the levels of piliation. Furthermore, the pRG5::pilA strain also killed WTP+ and WTP− controls did not remove U(VI) signifi- had a defect in outer membrane, heme-containing proteins (Fig. cantly (0.02 ± 0.04 and 0.05 ± 0.02 mM, respectively). X-ray S5), yet cells had very little U deposition in their cell envelope absorption near-edge structure (XANES) spectroscopy confirmed (Fig. S4). This finding is consistent with the pili functioning as the the reductive nature of the U removal activity and measured an primary site for U reduction. average of 70–85% U(IV) in all samples (Fig. 1A). Furthermore, the expression of the pilA gene relative to the internal control recA X-Ray Absorption Fine Structure (EXAFS) Analyses Demonstrate the did not change during the assay (Fig. S1), thus ruling out any de Reduction of U(VI) to Mononuclear U(IV). ULIII-edge EXAFS novo pilin expression. The extent of U(VI) removal corresponded spectra were modeled to determine the atomic coordination about well with the levels of piliation, which were measured as the U and characterize the U(IV) product in all of the strains (23). protein content of purified PilA-containing pili samples (Fig. S2). Models for the EXAFS spectra included signals from neighboring The pRG5::pilA piliation (3.6 ± 1.7 μg pili/OD600) was 2.5-fold P, U, and Fe atoms, but only C neighbors were found to accurately higher than WTP+ (1.5 ± 0.1 μg/OD600), which matched well with reproduce the measured spectra. The spectra were best described its superior capacity to remove U(VI) from solution (1.8 ± 1.0- by a mixture of U(IV) and U(VI) coordinated by C-containing − − fold higher than WTP+). By contrast, WTP− and PilA samples ligands. Only the PilA mutant required an additional P ligand. A had no detectable pili protein and reduced less U(VI). U signal corresponding to the U–U distance in uraninite at 3.87 Å The location of the U reductase system was studied by exam- was tested but was inconsistent with the measured spectra. Fig. ining the cellular localization of the U deposits in unstained whole 2A shows the magnitude of the Fourier-transformed spectra and cells by transmission electron microscopy (TEM; Fig. 1 B–E). The models for each spectrum. Fig. 2 B and C show, as examples, the piliated strains, WT and pRG5::pilA, preferentially deposited contribution of each path in the model in the real part of the P+ − Insets the U extracellularly and in a monolateral fashion, consistent with Fourier transform for the WTP+ and PilA cells, and Fig. 2 the localization of Geobacter’s conductive pili to one side of the show a molecular moiety of the U(IV) atomic environment that is cell (19). The pili filaments were interspersed with the dense consistent with the measured EXAFS. The WTP+ model includes deposits (Fig. S3). Elemental composition analyses of the pili- one C ligand bound to two O atoms of U in a bidentate fashion associated deposits by TEM energy dispersive spectroscopy and followed by a distant C atom (C3), and another C ligand (EDS) in the WTP+ confirmed the presence of U (Fig. S3). In bonded to one O atom of U(IV) in a monodentate fashion and contrast, extracellular U mineralization in the nonpiliated strains, attached to a distant O atom (Odist). This model was simulta- − neously refined to all spectra and was insufficient to reproduce WTP− and PilA , was limited to the cell surface and membrane − fi the PilA spectrum, which required an additional monodentate P vesicles. TEM thin sections of the unstained cells con rmed the 2 presence of extracellular, needle-like U deposits in the piliated ligand (Fig. 2C). The distances and σ values used to model the strains as well as discreet regions of U deposition on the outer spectra are listed in Table S1. The coordination numbers (Table S2) are consistent with 1–2 bidentate C ligands and two mono- membrane (Fig. S4). Only a few cells (8 ± 3% of the WTP+ and <1% of the pRG5::pilA) had periplasmic mineralization. Outer dentate C ligands per U atom. The number of Oax atoms (Noax) was also used to estimate the amount of U(IV) in these samples, as membrane foci of U deposition were also noticeable in the WTP−, but more cells (37 ± 13%) had periplasmic deposition. The in- there are two Oax atoms for each U(VI) atom and none for U(IV) – creased periplasmic mineralization in the WT − cannot be attrib- (23). An average of 3 4 replicates for each strain gives the values of P ± ± ::pilA ± uted to a differential expression of outer membrane c-cytochromes, 72 16% (WTP+), 81 6% (pRG5 ), 85 5% (WTP−), and ± − because outer membrane protein fractions had the same heme 76 10% (PilA ); this provides additional evidence that although − the extent of U(VI) removal depended on the expression of the profile and content as the WTP+ (Fig. S5). By contrast, the PilA mutant was partially defective in outer membrane c-cytochrome pili, the ability of the cells to reduce U(VI) to U(IV) did not. production (Fig. S5) and had the highest levels of periplasmic mineralization (85 ± 12% of the cells; Fig. S4), which suggests that U Reduction via Pili as a Protective Cellular Mechanism. The reverse correlation between piliation and periplasmic mineralization sug- gested that the pili-mediated reduction prevented U from per- meating and being reduced in the periplasm, thus preserving vital 0.6 A functions of the cell envelope. To test this, we used the fluorogenic U(VI) ’ U(IV) RedoxSensor Green dye to measure the cell s reductase activity (mostly, respiratory activity) (24) of the strains after U exposure in 0.4 reference to unexposed controls. The respiratory activity or “vi- tality” remaining after U exposure was higher in the piliated strains 0.2 and proportional to the levels of piliation (pRG5::pilA > WTP+; Fig. 3A). Inasmuch as the activity of the electron transport chain

Uranium removal (mM) is a vital function of the cell, these results suggested that the pili- 0 - ::pilA ’ WTP+ WTP- PilA pRG5 catalyzed reduction of U also preserved the cell s viability. To test this, we recovered the resting cells in growth medium and studied B C DE the cell’s survival (defined as the cell’s ability to maintain its in- tegrity and undertake division) (25) after exposure to U as a function of the length of the lag phase (Fig. 3B). Though cells that had not been exposed to U recovered rapidly and simultaneously, the strains exposed to U recovered in a step-wise fashion. The lag Fig. 1. Reduction of U(VI) to U(IV) (A) and TEM images of unstained whole phase was shortest (w18 h) in the hyperpiliated pRG5::pilA cells, w w cells showing the subcellular localization of the U deposits in the WTP+ (B), followed by the WTP+ ( 56 h) and the WTP− ( 81 h), and − WTP− (C), PilA (D), and pRG5::pilA (E) strains. (Scale bar, 0.5 μm.) correlated well with the levels of periplasmic mineralization of the

2of5 | www.pnas.org/cgi/doi/10.1073/pnas.1108616108 Cologgi et al. Downloaded by guest on October 3, 2021 AB C

WTP-

WTP+ PilA- pRG5::pilA

R (Å)

Fig. 2. ULIII-edge EXAFS spectra (symbols) and models (line). (A) Magnitude of Fourier transform spectra are offset for clarity. (B and C) Real part of Fourier − transform of WTP+ (B) and PilA (C). The components of the model are shown offset beneath the total model. (B and C Insets) U(IV) moiety consistent with the measured EXAFS spectra. U(IV), light gray; O, red; C, black; P, dark gray.

− strains (R2 = 0.947). The PilA mutant recovery was similar to the energy for growth from the reduction of Fe(III) oxides (6, 27), other nonpiliated strain, WTP−, yet more variable (lag phases a process that requires the expression of the conductive pili (19). ranging from 72 to 82 h). It also grew faster (w9 h doubling time We used a temperature-dependent regulatory switch (SI Materials compared with w11 h for the WT and pRG5::pilA strains) than the and Methods) to produce WT controls (WTP−) that did not as- other strains, which is expected to accelerate recovery. Despite semble pili, yet had WT levels and profiles of outer membrane these differences, the survival rates (calculated as the reverse of cytochromes. The lack of pili in the WTP− strain significantly di- the length of the lag phase) of all of the strains followed a linear minished the cell’s ability to remove U(VI) from solution, in- regression (R2 = 0.908) with the levels of pili protein. creased the degree of periplasmic mineralization, and reduced the respiratory activity of the cell envelope and the cell’s viability. SCIENCES ENVIRONMENTAL Discussion WTP− cells also had extensive outer membrane vesiculation, a Physiological Relevance of the Extracellular Reduction of U by process linked to the selective detoxification of unwanted peri- Geobacter’ plasmic materials by cells undergoing cell envelope stress (28). s Pili. Our results show that piliated cells immobilized − a greater amount of U and also prevented it from permeating in- Similarly, the inability of a PilA mutant to produce pili impaired side the periplasm, where it would have otherwise been reduced the yields of U reduction. This mutant strain also had a reduced nonspecifically by c-cytochromes and other low-potential electron outer membrane cytochrome content and, as a result, more U donors (17). As a result, the extracellular reduction of U via pili traversed the outer membrane and precipitated in the periplasm. also preserved the vital functions of the cell envelope and the cell’s The fact that the pili of G. sulfurreducens catalyze the extracellular viability. This mechanism is consistent with field studies showing reductive precipitation of U under physiological conditions con- that the indigenous Geobacter community that is stimulated during ducive to growth, is also in agreement with early studies with G. in situ bioremediation is metabolically active (9, 26) and gains metallireducens suggesting that the reduction of U is extracellular (10) and coupled to cell growth (1). In these studies (1, 10), cells were grown with Fe(III) citrate as an electron acceptor, which are culture conditions that promote pili expression in G. metal- A 0.4 lireducens (29) but not in G. sulfurreducens (19). Thus, the extra-

) G. - 0.3 cellular precipitation and sustained removal of U reported for /U + metallireducens is consistent with pili catalyzing the reaction as well. 0.2 Reduction of U to Mononuclear U(IV) Phases. Despite differences in

Vitality (U Vitality 0.1 the mechanism and yields of U reduction, the strains with the ::pilA 0 lowest levels of periplasmic mineralization (WTP+, pRG5 , - ::pilA WTP+ WTP- PilA pRG5 and WTP−) produced similar U LIII-edge EXAFS spectra that were modeled as mostly U(IV) coordinated by C-containing B 1 ligands in bidentate and monodentate fashion and that lacked any

) Fe- or P-containing ligands. The bidentate C1–C3 ligand is likely 600 biological in nature, as reported for the carboxyl coordinations OD

10 involving amino acids and lipolysaccharide sugars (30–32). In − 0.1 contrast, the PilA mutant, which had the highest degree of periplasmic mineralization, required an additional monodentate P ligand. This signal was small, with a coordination number of Growth (log ± 0.01 0.5 0.3, indicating that, on average, 50% of the U atoms con- 0 48 96 144 tained a P ligand, and the other 50% shared the atomic coordi- Time (hours) nation of the other strains. Alternative interpretations such as 25% or 12.5% of U atoms with two or four P ligands, respectively, Fig. 3. Effect of U(VI) exposure on cell vitality (A) and viability (B). (A)Vi- are unlikely, because the coordination number values for the C1 tality was measured as bacterial reductase (respiratory) activity with the and C2 signals did not decrease proportionally (50% and 75%, RedoxSensor dye in resting cells of the pili-expressing (WTP+ and pRG5::pilA) − respectively). It is also unlikely that the low levels of U(VI) re- and nonexpressing (WTP− and PilA ) strains and expressed as the ratio of − relative fluorescence units emitted by cells incubated with (U+) or without duced by the PilA strain contributed to the distinct spectra, be- − − (U )U.(B) Growth recovery of resting cells of the pRG5::pilA (black), WTP+ cause WTP cells reduced less U(VI) and did not require a P (dark gray), and WT − (light gray) after 6 h of U exposure (circles) in com- ligand for U coordination. The P coordination and the generalized P − parison with controls without U (lines). periplasmic mineralization observed in the PilA mutant cells

Cologgi et al. PNAS Early Edition | 3of5 Downloaded by guest on October 3, 2021 suggest that U(VI) permeated deep and fast into the cell envelope, activity of conserved components of Geobacter’s pilus apparatus where it formed carboxyl and phosphoryl-coordinated complexes to assess the effectiveness of in situ bioremediation schemes. The with periplasmic proteins and the peptidoglycan layer (30, 31) and possibility that conductive appendages such as the pili of Geobacter membrane phospholipids (33), respectively. are a widespread mechanism for U reduction also warrants special The formation of a mononuclear U(IV) phase has also been attention. The production of conductive appendages has been reported for other bacteria of relevance to U bioremediation (34, demonstrated in another U-reducing bacterium, onei- 35), yet contrasts with earlier reports of uraninite formation by densis (50). Furthermore, nanowire-mediated electrical currents Geobacter spp. (10, 36). The chemical composition of the me- have been proposed to couple spatially separated geochemical dium can influence the nature of the reduced U mineral (34). We processes in sediments (51). The extracellular needle-like U de- used a bicarbonate buffer and conditions used in previous studies posits observed in TEM thin sections of the piliated strains of with G. sulfurreducens (13), whereas studies reporting uraninite G. sulfurreducens (Fig. S4) also resembled the uraninite structures formation used PIPES-buffered solutions (36) or bicarbonate- of Desulfovibrio desulfuricans biofilms (52), which some authors buffered uncontaminated groundwater (10). Evidence for the have suggested represent mineralized nanowire-like appendages microbial reduction of U(VI) to nonuraninite U(IV) products is (17). Thus, the contribution of microbial nanowires to U reduction also emerging from field-scale studies (35, 37), whereas uraninite may be significant and, therefore, relevant for the optimization of in formation has been linked to conditions of reduced bioreducing situ bioremediation strategies. activities (38, 39); this suggests that abiotic factors may con- tribute to the formation of uraninite. Materials and Methods Strains and Culture Conditions. WT G. sulfurreducens PCA (ATCC 51573), − Model for the Reduction of U(VI) by Geobacter Bacteria. The direct a pilin-deficient mutant (PilA ) (19), and its genetically complemented strain correspondence observed between piliation, extent of U(VI) re- (pRG5::pilA) (19) were grown in fresh water (FW) medium supplemented duction, cell envelope respiratory activities, and cell viability sup- with 15 mM acetate and 40 mM fumarate. All cultures were incubated at port a model in which the conductive pili function as the primary pili-inducing temperatures (25 °C), except for the nonpiliated WTP− controls, mechanism for U reduction and cellular protection (Fig. S6). Pili which were grown at 30 °C (SI Materials and Methods). can reach several micrometers in length, thereby increasing the redox-active surface area available for binding and reducing U(VI) U(VI) Resting-Cell Suspension Assays. Resting-cell suspensions were prepared outside the cell. Although most of the U reduced by the piliated as described elsewhere (13), except that cells were harvested from mid–log- phase cultures (OD600,0.3–0.5) and resuspended in 100 mL reaction buffer cells was extracellular and associated with the pili, discreet regions fi of the outer membrane also participated in the reduction of U. In with 20 mM sodium acetate to a nal OD600 of 0.1. Heat-killed controls were G. sulfurreducens, most of the redox activity of the outer membrane prepared by autoclaving the cultures for 30 min. Suspensions were in- c cubated for 6 h at 30 °C with 1 mM uranyl acetate (Electron Microscopy is provided by abundant -cytochromes that decorate the cell sur- μ fi Sciences), as previously described (13). After incubation, 500- L samples were face as de ned foci (40). Thus, they could provide a mechanism for withdrawn, filtered (0.22-μm Millex-GS filter; Millipore) to separate the cells, reducing U in localized regions of the membrane and prevent it fi μ − − acidi ed in 2% nitric acid (500 L), and stored at 20 °C. All procedures were from permeating into the periplasm. In support of this, the PilA performed anaerobically inside a vinyl glove bag (Coy Labs) containing a H2: mutant cells, which had reduced outer membrane cytochrome CO2:N2 (7:10:83) atmosphere. The amount of U(VI) removed from solution content, preferentially reduced U in the periplasm. Some of the was estimated from the initial and final concentration of U(VI) measured in most abundant metalloproteins on the outer membrane of G. the acidified samples using a platform inductively coupled plasma mass sulfurreducens also are loosely bound to and easily detach from the spectrometer (Micromass, Thermo Scientific). membrane (40–42), providing a natural mechanism for releasing the U deposits. Some of these cytochromes also may be anchored X-Ray Adsorption Spectroscopy (XAS) Analyses. Resting cells incubated with U to a recently identified exopolysaccharide matrix (43), which may for 6 h were harvested by centrifugation (13,000 × g, 10 min, 4 °C) and loaded promote the extracellular reduction of U. Furthermore, although into custom-made plastic holders, triply packaged in Kapton film and sealed some areas of the outer membrane were voided of U reductase with Kapton tape (DuPont) under anaerobic conditions. Samples were stored at −80 °C and kept frozen during XAS measurements, which were performed activity, U(VI) was effectively prevented from permeating into the fl periplasm. The outer leaflet of the outer membrane of Gram- with a multielement germanium detector in uorescence mode, using the Pacific Northwest Consortium Collaborative Access Team beamline (sector 20- negative bacteria is mostly composed of LPS, which acts as an ef- BM) at the Advanced Photon Source (Argonne National Laboratory) and ficient permeability barrier against soluble toxic compounds (44). G. sulfurreducens standard beamline parameters, as described elsewhere (37). XANES meas- produces a rough LPS, i.e., it is composed of lipid urements were used to calculate the relative amount of U(VI) to U(IV) by A and a core oligosaccharide but lacks the O antigen (45). The core linear combination fitting of the spectrum with U(VI) and U(IV) standards. The oligosaccharide is the most highly charged region of the LPS and is spectra were energy aligned by simultaneously measured uranyl nitrate stabilized by metallic cations (46). Models suggest that rough LPS standards. EXAFS analyses are described in SI Materials and Methods. preferentially chelates and immobilizes uranyl ions over other ions (32) and produces carboxyl and hydroxyl coordinations (32), which Transmission Electron Microscopy. After U exposure, resting cells were is consistent with the C and O coordinations modeled from the adsorbed onto 300-mesh carbon-coated copper grids (Ted Pella), fixed with EXAFS spectra. These findings suggest that the rough LPS of G. 1% glutaraldehyde for 5 min, and washed three times with ddH2O for 2 min. sulfurreducens also functions as a protective barrier to prevent Unstained cells were directly imaged with a JEOL100CX operated at a 100-kV U(VI) from penetrating in the cell envelope. accelerating voltage. Thin sections for TEM were prepared as described in SI Materials and Methods. Implications for the in Situ Bioremediation of U. Insufficient knowl- edge of the biological mechanisms of contaminant transformation Vitality and Viability Assays. The RedoxSensor Green vitality assay (Invitrogen) often limits the performance of in situ subsurface bioremediation was used to measure the cell’s vitality (broadly defined as the levels of ac- fi Geo- tivity of a cell’s vital reactions) remaining after U exposure and in reference and long-term stewardship strategies. The identi cation of fl bacter’s pili as their primary U reductase provides a much-needed, to controls not exposed to U. This reagent yields green uorescence when Geo- modified by the bacterial reductases, which are mostly located in the elec- fundamental mechanistic understanding of U reduction by tron transport system of the cell envelope (24, 53). Resting cells were har- bacter spp. required to design effective in situ bioremediation × μ Geobacteraceae vested in a microcentrifuge (12,000 g), washed, and resuspended in 100 L strategies. Analyses of transcript abundance for key Tris-buffered saline (TBS) before mixing it with an equal volume of a work- genes are useful tools to predict the metabolic and physiological ing concentration of the dye. Fluorescence was measured in two biological state of Geobacter bacteria during in situ bioremediation (26, 47– replicates, with two technical replicates each, using a SpectraMax M5 plate 49), yet provide no information about the mechanism of U bio- reader (Molecular Devices) with an excitation of 490 nm and emission of 520 reduction. However, similar tools could be applied to monitor the nm. Cell viability after U exposure in comparison with controls without U

4of5 | www.pnas.org/cgi/doi/10.1073/pnas.1108616108 Cologgi et al. Downloaded by guest on October 3, 2021 was assayed by recovering the resting cells in NB medium with acetate and assistance during different phases of this work. This research was supported fumarate (NBAF) and measuring the length of the lag phase, as described by National Institute of Environmental Health Science’s Superfund Grant R01 previously (54). Before inoculation, resting-cell suspensions were gassed for ES017052-03 and US Department of Energy (DOE) Office of Science Office of 15 min with filter-sterilized air to reoxidize the U deposits (13) without Biological and Environmental Research Grant DE-SC0000795 (to G.R.). Sup- compromising G. sulfurreducens viability (54). Cells were harvested by cen- port was also provided by a Michigan State University College of Natural Science Hensley Fellowship and a Biogeochemistry Environmental Research trifugation (1,200 × g, 5 min), resuspended in 1 mL of wash buffer (final Initiative Fellowship (to D.L.C.). Pacific Northwest Consortium X-ray Sciences OD600 of 0.4), and mixed with 10 mL of NBAF in pressure tubes. The cultures Division facilities and research at the APS are supported by the US DOE Basic were incubated at 25 °C, and growth was periodically monitored as OD600. Energy Sciences, a Major Resources Support grant from Natural Sciences and Engineering Research Council, the University of Washington, Simon Fraser ACKNOWLEDGMENTS. We thank Matthew Marshall, Evgenya Shelobolina, University, and the APS. The APS is an Office of Science User Facility oper- Kazem Kashefi, Alicia Pastor, XouDong Fan, Blair Bullard, and the Advanced ated for the US DOE’sOffice of Science by Argonne National Laboratory and Photon Source (APS) and Sector 20 staff at Argonne National Laboratory for supported by US DOE Contract DE-AC02-06CH11357.

1. Lovley DR, Phillips EJP, Gorby YA, Landa ER (1991) Microbial reduction of uranium. 29. Childers SE, Ciufo S, Lovley DR (2002) Geobacter metallireducens accesses insoluble Fe Nature 350:413e416. (III) oxide by chemotaxis. Nature 416:767e769. 2. Sanford RA, et al. (2007) Hexavalent uranium supports growth of Anaeromyxobacter 30. Barkleit A, Moll H, Bernhard G (2009) Complexation of uranium(VI) with peptido- dehalogenans and Geobacter spp. with lower than predicted biomass yields. Environ glycan. Dalton Trans (27):5379e5385. Microbiol 9:2885e2893. 31. Benavides-Garcia MG, Balasubramanian K (2009) Structural insights into the binding 3. Lovley DR (2001) Bioremediation. Anaerobes to the rescue. Science 293:1444e1446. of uranyl with human serum protein apotransferrin structure and spectra of protein- 4. Anderson RT, et al. (2003) Stimulating the in situ activity of Geobacter to uranyl interactions. Chem Res Toxicol 22:1613e1621. remove uranium from the groundwater of a uranium-contaminated aquifer. Appl 32. Lins RD, Vorpagel ER, Guglielmi M, Straatsma TP (2008) Computer simulation of Environ Microbiol 69:5884e5891. uranyl uptake by the rough lipopolysaccharide membrane of Pseudomonas aerugi- 5. Istok JD, et al. (2004) In situ bioreduction of technetium and uranium in a nitrate- nosa. Biomacromolecules 9:29e35. contaminated aquifer. Environ Sci Technol 38:468e475. 33. Koban A, Bernhard G (2007) Uranium(VI) complexes with phospholipid model com- 6. Chang YJ, et al. (2005) Microbial incorporation of 13C-labeled acetate at the field pounds—a laser spectroscopic study. J Inorg Biochem 101:750e757. scale: Detection of microbes responsible for reduction of U(VI). Environ Sci Technol 39: 34. Fletcher KE, et al. (2010) U(VI) reduction to mononuclear U(IV) by Desulfitobacterium 9039e9048. species. Environ Sci Technol 44:4705e4709. 7. North NN, et al. (2004) Change in bacterial community structure during in situ bio- 35. Bernier-Latmani R, et al. (2010) Non-uraninite products of microbial U(VI) reduction. stimulation of subsurface sediment cocontaminated with uranium and nitrate. Appl Environ Sci Technol 44:9456e9462. Environ Microbiol 70:4911e4920. 36. Sharp JO, et al. (2009) Structural similarities between biogenic uraninites produced by 8. Wu WM, et al. (2006) Pilot-scale in situ bioremedation of uranium in a highly con- phylogenetically and metabolically diverse bacteria. Environ Sci Technol 43: taminated aquifer. 2. Reduction of u(VI) and geochemical control of u(VI) bio- 8295e8301. SCIENCES

availability. Environ Sci Technol 40:3986e3995. 37. Kelly SD, et al. (2008) Speciation of uranium in sediments before and after in situ ENVIRONMENTAL 9. Wilkins MJ, et al. (2009) Proteogenomic monitoring of Geobacter physiology during biostimulation. Environ Sci Technol 42:1558e1564. stimulated uranium bioremediation. Appl Environ Microbiol 75:6591e6599. 38. Kelly SD, Hesterberg D, Ravel B (2008) Analysis of soils and minerals using X-ray ab- 10. Gorby YA, Lovley DR (1992) Enzymatic uranium precipitation. Environ Sci Technol 26: sorption spectroscopy. Methods of Soil Analysis. Part 5—Mineralogical Methods, eds 205e207. Ulery AL, Drees LR (Soil Sci Soc Am, Madison, WI), pp 367e463. 11. Coppi MV, Leang C, Sandler SJ, Lovley DR (2001) Development of a genetic system for 39. Kelly SD, et al. (2010) Uranium transformations in static microcosms. Environ Sci . Appl Environ Microbiol 67:3180e3187. Technol 44:236e242. 12. Lloyd JR, et al. (2002) Reduction of actinides and fission products by Fe(III)-reducing 40. Qian X, Reguera G, Mester T, Lovley DR (2007) Evidence that OmcB and OmpB of bacteria. Geomicrobiol J 19:103e120. Geobacter sulfurreducens are outer membrane surface proteins. FEMS Microbiol Lett 13. Shelobolina ES, et al. (2007) Importance of c-Type cytochromes for U(VI) reduction by 277:21e27. Geobacter sulfurreducens. BMC Microbiol 7:16. 41. Mehta T, Coppi MV, Childers SE, Lovley DR (2005) Outer membrane c-type cyto- 14. Kim BC, et al. (2005) OmcF, a putative c-type monoheme outer membrane cyto- chromes required for Fe(III) and Mn(IV) oxide reduction in Geobacter sulfurreducens. chrome required for the expression of other outer membrane cytochromes in Geo- Appl Environ Microbiol 71:8634e8641. bacter sulfurreducens. J Bacteriol 187:4505e4513. 42. Mehta T, Childers SE, Glaven R, Lovley DR, Mester T (2006) A putative multicopper protein 15. Kim BC, Lovley DR (2008) Investigation of direct vs. indirect involvement of the c-type secreted by an atypical type II secretion system involved in the reduction of insoluble cytochrome MacA in Fe(III) reduction by Geobacter sulfurreducens. FEMS Microbiol electron acceptors in Geobacter sulfurreducens. Microbiology 152:2257e2264. Lett 286:39e44. 43. Rollefson JB, Stephen CS, Tien M, Bond DR (2011) Identification of an extracellular 16. Kim BC, Qian X, Leang C, Coppi MV, Lovley DR (2006) Two putative c-type multiheme polysaccharide network essential for cytochrome anchoring and biofilm formation in cytochromes required for the expression of OmcB, an outer membrane protein essential Geobacter sulfurreducens. J Bacteriol 193:1023e1033. for optimal Fe(III) reduction in Geobacter sulfurreducens. J Bacteriol 188:3138e3142. 44. Raetz CR, Whitfield C (2002) Lipopolysaccharide endotoxins. Annu Rev Biochem 71: 17. Wall JD, Krumholz LR (2006) Uranium reduction. Annu Rev Microbiol 60:149e166. 635e700. 18. Silhavy TJ, Kahne D, Walker S (2010) The bacterial cell envelope. Cold Spring Harb 45. Vinogradov E, Korenevsky A, Lovley DR, Beveridge TJ (2004) The structure of the core Perspect Biol 2:a000414. region of the lipopolysaccharide from Geobacter sulfurreducens. Carbohydr Res 339: 19. Reguera G, et al. (2005) Extracellular electron transfer via microbial nanowires. Na- 2901e2904. ture 435:1098e1101. 46. Schindler M, Osborn MJ (1979) Interaction of divalent cations and polymyxin B with 20. Butler JE, Young ND, Lovley DR (2010) Evolution of electron transfer out of the cell: lipopolysaccharide. Biochemistry 18:4425e4430. Comparative genomics of six Geobacter genomes. BMC Genomics 11:40. 47. Holmes DE, et al. (2008) Genes for two multicopper proteins required for Fe(III) 21. Strom MS, Lory S (1993) Structure-function and biogenesis of the type IV pili. Annu oxide reduction in Geobacter sulfurreducens have different expression patterns Rev Microbiol 47:565e596. both in the subsurface and on energy-harvesting electrodes. Microbiology 154: 22. Leang C, Qian X, Mester T, Lovley DR (2010) Alignment of the c-type cytochrome 1422e1435. OmcS along pili of Geobacter sulfurreducens. Appl Environ Microbiol 76:4080e4084. 48. Holmes DE, Nevin KP, Lovley DR (2004) In situ expression of nifD in Geobacteraceae in 23. Kelly SD, et al. (2005) Comparison of U valence state ratio determined from U L3-Edge subsurface sediments. Appl Environ Microbiol 70:7251e7259. XANES to EXAFS measurements. Advanced Photon Source Activity Report 2003 (Ar- 49. Holmes DE, et al. (2009) Transcriptome of growing in gonne Natl Lab, Argonne, IL). uranium-contaminated subsurface sediments. ISME J 3:216e230. 24. Gray D, Yue RS, Chueng CY, Godfrey W (2005) Bacterial vitality detected by a novel 50. Gorby YA, et al. (2006) Electrically conductive produced by She- fluorogenic redox dye using flow cytometry. Abstracts of the American Society of wanella oneidensis strain MR-1 and other microorganisms. Proc Natl Acad Sci USA Microbiology Meeting (Am Soc Microbiol, Washington, DC). 103:11358e11363. 25. Barer MR, Harwood CR (1999) Bacterial viability and culturability. Adv Microb Physiol 51. Nielsen LP, Risgaard-Petersen N, Fossing H, Christensen PB, Sayama M (2010) Electric 41:93e137. currents couple spatially separated biogeochemical processes in marine sediment. 26. Holmes DE, et al. (2005) Potential for quantifying expression of the Geobacteraceae Nature 463:1071e1074. citrate synthase gene to assess the activity of Geobacteraceae in the subsurface 52. Marsili E, et al. (2005) Uranium removal by sulfate reducing biofilms in the presence and on current-harvesting electrodes. Appl Environ Microbiol 71:6870e6877. of carbonates. Water Sci Technol 52:49e55. 27. Finneran KT, Anderson RT, Nevin KP, Lovley DR (2002) Potential for Bioremediation of 53. Kalyuzhnaya MG, Lidstrom ME, Chistoserdova L (2008) Real-time detection of actively uranium-contaminated aquifers with microbial U(VI) reduction. Soil Sediment Con- metabolizing microbes by redox sensing as applied to methylotroph populations in tam 11:339e357. Lake Washington. ISME J 2:696e706. 28. McBroom AJ, Kuehn MJ (2007) Release of outer membrane vesicles by Gram-negative 54. Lin WC, Coppi MV, Lovley DR (2004) Geobacter sulfurreducens can grow with oxygen bacteria is a novel envelope stress response. Mol Microbiol 63:545e558. as a terminal electron acceptor. Appl Environ Microbiol 70:2525e2528.

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